The objective of the proposed research is to improve tumor resection in fluorescence-guided surgery by improvement of selectivity and sensitivity of tumor visualization by a novel generation of fluorescence probes. There is an urgent need for methods that aid in the complete removal of tumor lesions during surgery, while minimizing resection of healthy tissue. Fluorescent real-time tumor visualization enables determination of the tumor margin and allows detection of sub-millimeter tumors, which are invisible by white light. Consequently, fluorescence-guided surgery reduces the probability of cancer recurrence and increases patient survival, compared to standard surgery. However, fluorescent probes used for tumor visualization have several properties that are not optimal: (1) The majority of probes provide a low tumor-to-background fluorescence ratio; (2) simultaneous targeting and visualization of multiple cancer markers is extremely difficult; and (3) distinguishing between tumors located on surface and in deep tissue is not possible. In this work, we will address these drawbacks through the development of fluorescent probes for further improvement in small-size tumor detection and differentiation between tumor and healthy tissue. The critical barrier in development of such probes is the lack of the suitable fluorophores. The optical properties of available fluorophores make it extremely difficult to simultaneously target multiple tumor markers. Moreover, currently available molecular fluorescent probes do not provide the ability to determine the depth of tumor localization in the tissue. Recently, we have demonstrated that a bacteriochlorin-galactosylated human serum albumin conjugate visualizes in vivo peritoneal ovarian cancer metastases with both great selectivity and great sensitivity. The high selectivity and sensitivity of this probe result from quenching of the bacteriochlorin fluorescence upon attachment to a protein, and fluorescence activation occurring only in the target cells. Moreover, we have shown that bacteriochlorin enables differentiation between tumors located on the surface and in deep tissue because of its ability to be excited by both green and near-IR light. In addition, bacteriochlorins exhibit exceptionally narrow emission bands, with wavelength tunability across the near-IR region (700-800 nm) by simple structural modifications, making them well-suited for multicolor simultaneous detection of multiple targets. Here we propose to develop a whole family of bacteriochlorin derivatives with fluorescence that is preferentially activated in the target cells. These derivatives will possess a common green and near-IR excitation wavelengths and distinctive, well resolved emission wavelengths that enable selective visualization of tumors located on the surface and in deep tissue and simultaneous targeting of multiple markers. Subsequently, the optimized fuorophores will be conjugated with targeting proteins, and the performance of the resulting probes in multicolor fluorescence-guided surgery will be determined in vivo. The following specific aims will be realized: (1) Development of a family of bacteriochlorins with target-specific activatable fluorescence. A family of bacteriochlorin derivatives, with distinctive emission bands spanning 700-800 nm, and a high ratio of fluorescence quenching upon conjugation to model proteins, and dequenching upon protease-induced protein degradation will be developed. (2) Development of a family of fluorophores for simultaneous targeting of multiple markers. To tailor bacteriochlorins developed in aim 1 to multicolor fluorescence-guided surgery, a family of their derivatives, with common excitation wavelengths in visible (500 nm) and near-IR (~675) regions and distinctive emission wavelengths, will be developed. A series of energy-transfer, bacteriochlorin-based arrays will be synthesized, with additional chromophores that strongly absorb at 500 nm (BODIPY) and 675 nm (chlorin). Suitable hydrophilic derivatives for attachment to proteins will be developed. (3) Development and assessment of fluorescent probes for multicolor detection of peritoneal ovarian cancer metastases. Two distinctive fluorophores developed in aim 2 will be conjugated to galactosylated human serum albumin and a trastuzumab antibody, respectively. The resulting conjugates will be used for two-color detection of ovarian cancer cells that over-express both lectin and epidermal growth factor. The sensitivity and specificity of two-color vs. one-color detection will be compared.